PROJECT DESCRIPTION Activation of myeloid differentiation pathways always accompanies blood regeneration after stress, the development of hematological malignancies and physiological aging. However, our understanding of what activate myelopoiesis in such deregulated conditions is still very limited. Our goals in this NHLBI OIA application are to (1) decipher the cellular and molecular mechanisms controlling emergency myelopoiesis pathways; (2) understand how the hijacking of these mechanisms contributes to deregulated hematopoietic stem cell (HSC) function and blood production in stress, disease and aging; and (3) identify novel targets for therapeutic interventions aimed at correcting blood production in these deregulated contexts. We recently showed that the output of the myeloid lineage at steady state reflects the differential production by HSCs of a small number of myeloid-biased multipotent progenitors (MPP), called MPP2 and MPP3, and a large number of lymphoid-biased MPPs, known as MPP4 or LMPPs, which both give rise to granulocyte/macrophage progenitors (GMP) and contribute to myelopoiesis (Pietras et al., 2015). During blood regeneration, we found that HSCs are transiently induced to overproduce MPP2/3 and that MPP4 are reprogrammed towards almost exclusive myeloid output, in large part due to cytokine stimulation and the triggering of specific regulatory pathways (Reynaud et al., 2011; Pietras et al., 2015; 2016). An important consequence of the activation of this myeloid regeneration axis is the formation of defined GMP clusters in the bone marrow (BM) cavity, which drive the local overproduction of granulocytes (Hrault et al., submitted). This newly identified process of GMP cluster formation is finely tuned by the timed release of important BM niche signals, and transient activation of an inducible self-renewal network in a subset of GMPs. Altogether, the remodeling of the MPP compartment and induction of GMP cluster formation represent novel and targetable mechanisms of emergency myelopoiesis, which are transiently activated during blood regeneration but are continuously triggered in myeloid malignancies. We are now interested in exploring the contribution of these mechanisms to other deregulated contexts such as inflammation and aging, and in answering an exciting set of new questions that have directly emerged from these studies. In particular, we would like to understand the molecular and cellular basis for the functional heterogeneity observed in the MPP and GMP compartments, map the mechanisms of HSC lineage commitment and their links to the pro-inflammatory BM milieu, and decipher the contribution of the biophysical properties of the BM niche to HSC and myeloid progenitor fate decisions. We also would like to conduct correlative studies with human cells and leukemic patient samples to establish whether aberrant activation of similar emergency myelopoiesis pathways contribute to deregulated blood production in humans. Taken together, these studies are paradigm shifting for understanding the mechanisms controlling blood regeneration and their deregulations in leukemia and aging, and for identifying new targets for translational applications and the treatment of a broad range of blood disorders in humans. While many current therapies treat blood disorders by targeting the malignant and/or overproduced blood cells, our objective is to identify new biological process upstream of these cells to treat blood disorders by using anti-HSC differentiation therapies and by restoring proper regulation of blood production.